【作者】 乔松林；

【导师】 张改平；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2009， 博士

【摘要】 Fc受体(FcR)为特异亲和免疫球蛋白(Ig) Fc片段的细胞表面分子,广泛表达于免疫细胞表面,具有许多重要的生理功能,是体液免疫与细胞免疫的联系纽带,在机体免疫调控中起关键作用。目前人和小鼠的三类IgG Fc受体(FcγRI、FcγRII和FcγIII)研究最为深入。本研究克隆和鉴定了猪IgG三类Fc受体;研究了PRRSV变异毒株和经典毒株感染后活化性Fc受体和抑制性Fc受体的转录动态差异及与免疫抑制的关系;构建了稳定表达猪FcγRII的Marc-145细胞系,研究了该受体在介导PRRSV ADE中的重要作用。用人CD64氨基酸序列检索NCBI的EST数据库(包括除人和老鼠以外的所有物种核苷酸序列)(tBLASTn),发现了6个猪的高同源overlapping序列,利用这些序列信息设计引物,采用RT-PCR从猪外周血白细胞中扩增到了猪FcγRI cDNA序列,命名为poFcγRI。poFcγRI ORF全长1038 bp,编码346氨基酸的糖蛋白。胞外区包括三个Ig样结构,有4个N-糖基化位点。poFcγRI和人、牛与老鼠的FcγRI在氨基酸水平上分别有79%、69%和57%的相似性。胞外环区相似性较高,分别为77%、74%和70%,这提示人、牛、猪、鼠的FcγRI和IgG的结合域高度保守。随后,RT-PCR检测到了poFcγRI mRNA在巨噬细胞、单核细胞中表达量较高,在多形核细胞中也有表达,在淋巴细胞中没有表达,和人与小鼠CD64的表达谱类似。将poFcγRI ORF基因亚克隆到真核表达载体pcDNA构建成重组质粒pcDNA/poFcγRI,再用脂质体法转染COS-7细胞,用玫瑰花环实验鉴定了poFcγRI配体亲和特性。对猪IgG Fc受体的进一步研究发现猪IgG Fc受体共由三类分子组成,即FcγRI、FcγRII和FcγIII,克隆了这三类受体cDNA,其中FcγRII胞内区有保守的ITIM基序,为抑制性受体;FcγRI和FcγRIII胞内区没有发现已知的信号传导基序,通过γ链的ITAM基序进行信号转导,因而这两个受体为活化性受体。发现FcγRII至少存在两个亚类,即FcγRIIB1和FcγRIIB2,FcγRIIB1胞内区有一插入序列,使胞内区由47个氨基酸延长到99个氨基酸。这两个亚类胞内区均存在ITIM基序,因此均为抑制性受体。活化型受体和抑制型受体共表达于免疫细胞表面,共同调节抗体对免疫细胞的活化作用。抗原抗体复合物和免疫细胞表面的活化型受体给合,能活化免疫效应细胞,诱发多种免疫学效应。抑制型受体的作用刚好相反,通过免疫复合物与抑制型受体交联,抑制活化受体介导的细胞活化。研究PRRSV感染后活化型和抑制型Fc受体的转录动态,对于揭示PRRSV感染引起的免疫抑制有重要意义。通过Real-time PCR检测不同毒株PRRSV感染后猪体活化性和抑制性Fc受体的转录动态,发现PRRSV感染后活化性受体FcγRI和FcγRIII均迅速下调,而抑制性Fc受体FcγRII感染后有轻微上调。PRRSV感染后机体能够快速诱导产生高水平的特异性抗体,但感染后活化性Fc受体下调导致抗体和抗原抗体复合物不能活化免疫效应细胞,从而引起细胞免疫功能下降,这是PRRSV感染引起免疫抑制的分子基础。抗体依赖增强作用(ADE)是PRRS防控中遇到的最大的难题之一。本研究将猪FcγRII基因转染Marc-145细胞,通过G418连续筛选和克隆获得了稳定表达猪FcγRII的Marc-145细胞系。用PRRSV BJ-4株与1:320稀释的抗PRRSV IgG感染克隆化的Marc-145细胞系,在有抗体存在的情况下PRRSV感染收获的病毒量明显高于对照组,证明了poFcγRII能够介导PRRSV ADE作用。在PRRSV免疫效果评价当中,现有的评价标准是测定疫苗免疫后的PRRSV特异性抗体。但ADE作用的存在使PRRSV特异抗体在特定情况下不仅没有保护作用,反而有助于感染。本研究构建的稳定表达猪FcγRII的Marc-145细胞系能够方便的评价抗体的ADE作用,从而为研制高效PRRSV疫苗提供技术支持。更多还原

【Abstract】 Fc receptors (FcR) are a group of crucial molecules expressed on the surface of immune cells, which bind the Fc region of immunoglobulins with specific affinities and have a number of important biological functions. FcRs play a critical role in immune regulation by providing a link between the humoral and cellular immune responses. Three classes of FcγR (FcγRI, II, III), present in human and mouse, are the best characterized. In this study, three classes of porcine FcγRs was cloned and characterized. The relationship between immune suppression and dynamic expression of porcine activation and inhibitory FcγRs after infection with PRRSV isolates was investigated. Porccine FcγRII was been proved playing a crucial role in mediation the antibody-dependent enhancement (ADE) of PRRSV infection by construction the Marc-145 cell line with stable poFcγRII expression.By screening a translated EST database (which is composed of sequences from specieces other than human and mouse) with the protein sequence of the human CD64,we identified six putative overlapping porcine homologues. With this sequence information,a pair of primers was designed and the full-length cDNA encoding porcine FcγRI we isolated (poFcγRI) from peripheral blood leucocyte RNA using RT-PCR. Porcine FcγRI contains an open-reading frame (ORF) of 1038 bp length, encoding a 346 amino acids glycoprotein. Extracellular domain of poFcγRI consists of three Ig-like domains and four N-glycosylation sites. The predicted amino acid sequence of whole poFcγRI was found to be 79%, 69% and 57% identitical with human, bovine and mouse counterpart; however, a comparison of the three extracellular domains alone between the four species shows a higher identity of 77%, 74% and 70% respectively. This is in agreement with the generally highly conserved nature of the individual FcR binding domains. In order to examine the cell distribution of poFcγRI, RT-PCR was performed on mRNA from selective cellular subsets. Porcine FcγRI is expressed in PAM, monocytes, and PMN. A higher abundance of transcripts was found in PAM and monocytes than in PMN, where only a trace transcript was found. Then, a cDNA for the complete coding region of poFcγRI was subcloned into the expression vector pcDNA3, and transfected into COS-7 cells. The COS-7 cells transfected with the poFcγRI cDNA were able to bind chicken erythrocytes sensitized with porcine IgG.Three classes of porcine FcγR, named FcγRI, FcγRII and FcγIII, was further characterized. Of which, poFcγRII belongs to inhibitory Fc receptor, as an ITIM motif was found in intracellular domain. Whereas, porcine FcγRII and FcγIII, devoid of know signal motif, seem to be activatory Fc receptors and that they may form an activation receptor complex withγchain on the surface of immune cells. Two isoforms of poFcγRII, FcγRIIB1 and FcγRIIB2 were further identified. FcγRIIB1 has an in-frame insertion, which increases the cytoplasmic region from 47 to 99 amino acids. The two isoforms have ITIM motif in the intracellular domain, so they both belong to the inhibitory receptors.The paired expression of activating and inhibitory molecules on the same cell is the key for the generation of a balanced immune response. After binding with the activating Fc receptor, immune cells can be activated and a variety of immunological effects were induced by antigen-antibody complex. The role of inhibitory receptors is exactly the opposite. Thus, it is important to study the activating and inhibitory Fc receptor transcription dynamicas after PRRSV infection for further invesitgaiton the immune suppression by PRRSV infection. Using real-time PCR, the activating and inhibitory Fc receptor transcription dynamic in porcine peripheral blood leucytes was investigated after infection with PRRSV isolates. The expression of activation receptor, FcγRI and FcγRIII, was found to be rapidly reduced, and the inhibitory Fc receptor, poFcγRII, had a slight increase after infection. PRRSV infection rapidly induced high levels of PRRSV-specific antibodies, but the the immune effector cells were not to be activated by antibody and antigen-antibody complex due to the decreased expression of activating FcγRs. This may be the molecular basis of immunosuppression after PRRSV infection.Antibody-dependent enhancement (ADE) is one of the major problems in prevention and control PRRS. In this study, poFcγRII gene was transfected to Marc-145 cells. After G418 selection and continuous cloning, construction of Marc-145 cell line with stable expression of poFcγRII was achieved. Then, Marc-145 cell lines with stable poFcγRII expression was infected with PRRSV BJ-4 strain and 1:320 dilution of anti-PRRSV IgG together or PRRSV alone. The harvested virus of Marc-145 cell lines infection with PRRSV and antibody together was significantly higher than that of PRRSV absence of antibody. Thus, poFcγRII mediated the ADE of PRRSV infection. The existing evaluation criteria for the effect of PRRSV vaccine is determined by detection of the PRRSV vaccine specific antibodies. As ADE of PRRSV infection, PRRSV-specific antibodies could not protect pigs from PRRSV infection and contribute to infection in some cases. In this study, Marc-145 cell lines with stable poFcγRII expression can be used for evaluation the ADE effect of antibodies, so it will provide technical support for develop ping efficient PRRSV vaccines.更多还原